Recently, due to high oil prices and environmental concerns, the microbial production of biofuels has received a great deal of attention. Also, biodiesel has been substituted for light oil or a mixture of biodiesel with light oil has emerged as an alternative fuel that can be used in diesel engines, and thus the market size of biodiesel has rapidly increased. In the European Union (EU) in 2008, 6.6 million tons of biodiesel was produced, which a market size of 5.5 billion euro (Biodiesel Market, Frost & Sullivan). Also, in USA in 2006, 3 billion gallons of biodiesel was produced (Biodiesel Market, Global Industry Analysts Inc, 2006. 5).
Biodiesel is advantageous in that it has a high burning rate and thus low emission of poisonous gases, an about 10% lower heating value than that of light oil, and a higher ignition point than that of light oil, indicating that it is more stable during storage and transport. Biodiesel has been mainly produced by processing the fatty components of animals and plant so as to have properties similar to those of light oils or allowing vegetable oils and fats (rice bran, waste cooking oil, soybean oil, rapeseed oil, etc.) to react with alcohol. However, in this case, there is a shortcoming in that it is difficult to produce biodiesel in large amounts. Thus, if biodiesel suitable as an alternative fuel for light oil is produced in large amounts using microorganisms, the import of crude oil will decrease and the emission of greenhouse gases will decrease, resulting in environmental effects.
Meanwhile, oil is an energy carrier that is synthesized and accumulated in microbial cells when microorganisms are rich in carbon sources but lack other growth factors (nitrogen, phosphorus, oxygen, sulfur, etc.). When the environment of microbial growth changes so that the other growth factors are supplied to microorganisms, the accumulated oil will be degraded and used as an energy source. It is known that oil can consist of more than 100 kinds of monomers depending on the kind of oil-producing microorganism, the kind of chemical material supplied, changes in culture conditions, etc.
Recently, the technology of producing fatty acid alkyl ester by adding alcohol to vegetable fatty acid such as a sugar cane was developed, and the produced fatty acid alkyl ester is currently being used as a biodiesel fuel. Also, methods for esterifying free fatty acids are disclosed in European Patent Publication No. 127104A, European Patent Publication No. 184740A and U.S. Pat. No. 4,164,506. According to the disclosures of these patents, an esterification reaction is carried out by heating a mixture of fatty acid and fatty acid triglyceride together with methanol. In addition, European Patent Publication No. 708813A discloses a method of producing fatty acid alkyl ester from oils and fats in an increased yield, in which free fatty acid is separated from a glycerin resulting from ester interchange and is then esterified.
However, in this method, it is difficult to obtain large amounts of fatty acid or free fatty acid. In addition, it is difficult to increase the accumulation and production of vegetable fatty acids that are currently most frequently used, because the growth period of plants is long and a metabolic engineering approach to produce the vegetable fatty acids is somewhat difficult.
Accordingly, the present inventors have made extensive efforts to develop a novel method capable of producing a fatty acid alkyl ester, which can be used as biodiesel, with high efficiency and productivity using a metabolic engineering approach, and as a result, have found that the fatty acid alkyl ester can be produced with high efficiency by maximizing the production of oil in oil-producing microorganisms using a metabolic engineering method, and then inducing the autolysis of the oil in the microorganisms to produce a free fatty acid which is then converted to an alkyl ester, thereby completing the present invention.